Preparation of microbial alkaline protease by fermentation with bacillus subtilis, variety licheniformis

ABSTRACT

Alkaline protease is prepared by culturing a selected microorganism of the species Bacillus subtilis, variety licheniformis in a nutrient medium optionally in the presence of cottonseed protein, and thereafter separating the alkaline protease thereby produced from said medium.

United States Patent Louis I. Feldman Spring Valley, N.Y. 4,737

Inventor Appl. No. Filed Patented Assignee Jan. 21, 1970 Nov. 30, 1971Baxter Laboratories, Inc. Morton Grove, Ill.

PREPARATION OF MICROBIAL ALKALINE PROTEASE BY FERMENTATION WITH BACILLUSSUBTlLlS, VARIETY LICHENIFORMIS 10 Claims, No Drawings US. Cl. 195/66 Rint. Cl ..Cl2d 13/10 Field oi Search 195/66, 62

References Cited OTHER REFERENCES Hall, The Proteolytic Enzymes ofBacillus licheniformis" Dissertation Abstracts Vol. 27 (1966) pages2277-8 to 2278- B.

Primary Examiner-Lionel M. Shapiro Attorneys-Walter C. Kehm and W.Garrettson Ellis ABSTRACT: Alkaline protease is prepared by culturing a,

PREPARATION OIv MICROBIAL ALKALINE PROTEASE BY FERMENTATION WITHBACILLUS SUBTILIS, VARIETY LICI'IENIFORMIS BACKGROUND OF THE INVENTIONThe term alkaline protease" stands for a class of proteolytic enzymeswhich are operative in the alkaline pH range, e.g., about pH 8 to 11.5.These materials show great promise as cleaning agents for removingstains from clothing in the frequent cases in which the stains haveprotein components. A further advantage of these materials is that theycan generally be used in conjunction with commercial detergents. Thus,detergent formulations are available which contain alkaline protease forimproving the performance of the formulations.

It is presently known to the art to produce alkaline protease byculturing the organism Bacillus subrilis. However, many organisms ofthis species give a low yield of alkaline protease, and thus theproduction of the material from these organisms is inefficient andexpensive.

DESCRlPTlON OF THE INVENTION It has been found that the licheniformisvariety of the organism Bacillus subtilis gives greatly improved yieldsof alkaline protease when compared with other varieties of Bacillussubtilis, and as such provides a substantial advantage in thepreparation of alkaline protease because of the substantial gains inproduction efficiency obtained by use of the organism. Licheniformis isconsideredbysome authorities to be a separate species from subtilis.However, it is very similar to subtilis, and is considered as asubspecies or variety thereof for purposes of this application.

The protease produced from licheniformis is separated from the culture,preferably by precipitation with methanol or another nonaqueous, polarsolvent. 7

The alkaline protease produced by this invention generally has greateractivity at higher temperatures than other alkaline proteasesfacilitating its use in automatic washing machines.

lt has further been found that the Bacillus subtilis, varietylichemfiirmis strain deposited at the Northern Regional ResearchLaboratories at Peoria, lll. under the number NRRL B3723 is uniquelyefiective to produce significantly higher yields of high qualityalkaline-stable protease having improved proteolytic activity at highertemperatures, and having a maximum proteolytic activity at about 57 to67 C., measured at pH 8.5. This organism, when cultured in accordancewith this invention, gives unsurpassed yields of superior alkalineprotease.

Lichenifonnis organisms are cultured in a nutrient medium, preferablywith periodic incremental additions of soluble carbohydrate during amajor portion of the culturing period to maintain a reducing sugar levelof about 0.4 to 1 weight percent of the culture medium, calculated asglucose. Preferably, the culturing is performed under aerobic conditionsat a temperature of 30 to 40 C. and at a pH of 5.0 to 8.0 throughout theentire period of culturing in order to obtain an advantageously highyield of alkaline protease.

One advantage of the use of Iicheniformis is that it gives a low yieldof amylase byproduct, while other high yield protease-producingorganisms tend to produce large amounts of amylase.

It is particularly preferred to maintain a pH of 6.0 to 6.6 and atemperature of 34 to 38 C. throughout the culturing step.

It is also desirable for the nutrient medium to contain a small amount(e.g., about 2 to 5 weight percent) of cottonseed protein, which raisesthe production of alkaline protease to a particularly high level.

After the culturing has proceeded, generally for a period of time toproduce a maximum yield of alkaline protease, the protease is typicallyseparated from the rest of the medium. This can be done by filtering themedium to remove extraneous solids, washing the solids, and thenprecipitating alkaline protease from the filtrate.

Polar solvents having a low water content are generally useful toprecipitate the alkaline protease from a culture filtrate. Typicalsolvents which can be used are acetone, diethylether, and alcohols of nomore than about three carbon atoms such as methanol, ethanol andisopropanol. Mixtures of polar solvents can also be used. Another classof materials for precipitating alkaline protease from a culture filtrateare the soluble salts, for example, ammonium sulfate, sodium sulfate,potassium nitrate, and sodium chloride.

Prior to precipitation of alkaline protease from the filtrate of theculture, it is desirable to evaporate the culture filtrate and addedwash solution to about 30 to 40 percent of its initial weight. Thealkaline protease is then precipitated from this evaporate, preferablywith an alcohol having no more than three carbon atoms such as methanol.

The protease so produced generally has a maximum proteolytic activity ina temperature range of about 57 to 67 C. at about pH 8.5.

The following example is for illustrative purposes only and is not to beconstrued as limiting the invention of this application.

EXAMPLE 1 Fermentations were conducted in 1 liter, baffled Erlenmeyerflasks containing 200 ml. of the following medium:

Weight Percent Cornstarch degraded with alpha amylase,

enzyme Lactose Cottonseed protein (Pharrnamedia) manufactured byTrader's Protein Division, Fort Worth, Tex. 3

The above mixture was neutralized to a pH of 6.7 with ammoniumhydroxide. The mixture was then sterilized at about 121 C. in anautoclave at an overpressure of one atmosphere for about 15 minutes.

Several flasks prepared in the manner described above were inoculated,each with a separated strain of either Bacillus subrilis or Bacillussubtilis, variety lichemfarmis. The flasks were then incubated on arotary shaker at 37 C. for 138 hours. After this the concentration ofalkaline protease in the resulting mixture was determined in thefollowing manner:

A bufi'er solution of 3.0 grams of tris-(hydroxymethyl) aminomethane(known as Tris), mixed with 400 ml. of distilled water is prepared andtitrated with l N Hydrochloric acid to pH 8.5 while stirring. Distilledwater is then added to make a volume of 500 ml.

A substrate solution is made by adding 1.3 grams of casein to ml. of a0.05 M "Tris" buffer at pH 7.6 and stirred with heating to dissolve thecasein. After cooling, additional buffer solution is added to make avolume of ml.

A mixture to which the enzyme-containing products prepared in the abovefermentation is added is made in a series of test tubes by mixing ineach tube 3 ml. of the above substrate solution, 3 ml. of 0.02 N sodiumhydroxide solution, and 3 ml. of 0.044 M sodium tripolyphosphatesolution brought to pH 8.5 with HCl. The tubes are closed with stoppersand brought to a temperature of 37 C. The pH should be 8.5 or above.

A watch with a second hand is consulted, and at a given time, 3 ml. ofan enzyme solution containing the alkaline protease to be assayed isadded to each test tube, except to a control substrate blank," whichinstead receives 3 ml. of the buffer solution prepared above. Tubes aremixed for at least 30 seconds by tapping the tube walls and thenincubating at 37 C. Exactly 15 minutes after adding the enzyme solution,there is added to each tube grams of trichloroacetic acid solutionprepared by adding 18 grams of trichloroacetic acid, 19 grams of sodiumacetate and 18.9 ml. of glacial acetic acid to a container, and thendiluted to 1 liter with distilled water. This mixture destroys theenzyme and prevents further hydrolyzing by the enzyme of the caseinpresent. The contents of each tube are then filtered through 1 1 cm.Whatrnan No. 42 paper with the first portion of the filtrate beingrefiltered through the paper.

Simultaneously, an "enzyme blank" is prepared by incubating 3 ml. of theenzyme solution for minutes at 37 C. The solution is then added to amixture of 3 ml. each of substrate solution, 0.2 N sodium hydroxide, and0.044 M sodium tripolyphosphate after the mixture has been incubated for15 minutes at 37 C., and 10 ml. of the above trichloroacetic acidsolution added. After adding the enzyme solution, the mixture isincubated again at 37 C, with occasional shaking for onehalf hour andfiltered.

The optical densities of each of the above filtrates are then determinedat 275 p. using a 10 mm. cell and a spectrophotometer which is set withthe 100 percent transmission point being the reading of the substrateblank prepared above. The readings are then corrected by subtracting theop tical density of the enzyme blank" prepared above from the opticaldensities of the other test solutions.

The amount of alkaline protease present is then expressed in activityunits which are calculated by the following fonnula:

The corrected optical density at 275 mg, of the hydrolyzate mixture inthe Protease test tube Volume (i.e. 22 ml.) fi y The optical densityTime (i.e. 15 minutes) umts at 275 mg of 1.5

mg. tyrosine per 7 m]. (i.e. 0.0114) In short, one activity unit is thatamount of enzyme which produces in 1 minute, under the conditions of theabove test, enough tyrosine and other materials by hydrolyzing casein toprovide an optical density at 275 p. which is the same as a tyrosinesolution containing 1.5 mg. of tyrosine per ml.

Referring again to the fermentation samples prepared above, each of thesamples was assayed for the presence of protease activity units afterthe incubation period described above. The activity units present ineach sample are listed below. Each of the strains are available from theNorthern Regional Research Laboratories of Peoria, 111., and aredescribed by the culture numbers assigned them.

Species and Strain of Cultured Organisms Protease Activity Units Per ml.Produced After I38 Hours Fermentation Afier the 1 14th hour offermentation, culture NRRL B1716 had 5933 units, and culture NRRL B3723had 8890 protease activity units per ml. The remaining lichemjformi:strains had less protease activity than at 138 hours of fermentation.

In view of the above, it is plain that Iichemformir generally yields alarge increase of alkaline protease over the yield provided by othertypes of Bacillus subtilis, and NRRL B3723 gives about 50 percentgreater yield of protease activity than the best previously knownlichemformis strain.

The alkaline protease in the samples prepared above by fermentation withBacillus subtilis, variety licheniformis can be extracted by filtering,and precipitating impurities from the filtrate by adding methanol to a35 weight percent concentration and filtering again. The enzyme is thenprecipitated from the filtrate by adding more methanol to reach a finalmethanol concentration of percent, while holding the temperature below 4C. Particularly good results are obtained by adjusting the pH of themixture to 5.5 to 5.6 prior to the precipitation of impurities withmethanol.

EXAMPLE 2 A. A culture of Bacillus subtr'lis, variety lichenrj'ormtlsNRRL B3723 was heated at 60 C. for 20 minutes in a 0.85 percent sodiumchloride solution, inoculated onto a potato plug, and grown for 24 hoursat 37 C. Following this, portions of the potato plug were added to 1000ml. of a mixture containing 4 grams of dextrose, 5 grams of sodiumchloride, 10 grams of tryptone, 3 grams of beef extract, 1 gram ofK,HPO,, 3 grams of yeast extract, and the balance water. This mixturewas incubated for 18 hours at 37 C. on a rotary shaker to form abacterial inoculum.

A mash composition was prepared containing the following ingredients:

Parts by Weight trace trace Sufficient to make a total weight of 1,000parts by weight Hydrated manganese sulfate Hydrated zinc sulfate WaterThe above mash is batch sterilized for 45 minutes at to C., and cooledto 35 to 37 C. Sterile ammonia is added to provide a pH of 6.3 to 6.6.About 0.3 weight percent of the bacterial inoculum previously preparedis added to the mash, and fermentation is allowed to proceed for about20 hours with aeration and agitation. During this period the temperatureis maintained at 35 to 37 C., and the pH is maintained between 6.1 and6.5 by periodic addition of ammonia.

Following this, another mash composition similar to the above mash isprepared, except that 372 parts by weight of the 45 percentenzyme-degraded starch solution is added, with correspondingly lesswater being added. The mash is batch sterilized under the previouslydescribed conditions, cooled to 35 to 37 C., and ammonia added toprovide a pH of 6.4 to 6.6.

About 80 pounds of the previously fermented mash are added to the newmash composition, and fermentation is allowed to proceed for 58 hoursunder the previous fermentation conditions. The carbohydrate content ofthe fermentation mixture is periodically assayed, and periodic additionsof Protease Activity Hours of Fermentation Units per ml.

The resulting mash is then filtered and the filter residue is washedwith tap water.

The filtrate is evaporated to a concentration of about 18 Be. and isthen cooled to 3 C. and adjusted to a slightly acid pH. Methanol isadded to a methanol concentration of about 80 percent to precipitate theprotease, which is then filtered away from the liquid phase.

The alkaline protease produced by the above process exhibits a maximumproteolytic activity at about 60 to 65 C., measured by its hydrolysis ofcasein for minutes at pH 8.5. The alkaline protease exhibits maximumproteolytic activity at about pH 10 at a temperature of 37 C. and atabout pH 9.6 at 6 1 C.

B. The experiment of example 2(A) was again repeated except that theorganism of strain NRRL B3723 was replaced with another strain ofBacillus sublilis, variety licheniformis. The results were as follows:

Protease Activity Units EXAMPLE 3 The experiment of example 2(A) wasrepeated except that the corn steep liquor ingredient, and some water,was replaced with 3 weight percent of cottonseed meal '(Pharmamedia).The results were:

Protease Activity Units Hours of Fermentation Per ml.

EXAMPLE 4 Generally equivalent results to those of examples 2(A) areobtained upon (1) substitution of the 12.9 parts by weight of phosphoricacid with 3.2 parts by weight of disodium phosphate and 4 parts byweight of monosodium phosphate, or upon (2) substitution of the lactosewith an equivalent amount of water.

EXAMPLE 5 Generally equivalent results are obtained upon substitution ofthe last mash composition used in example 2(A) with a mash compositionof the following formulation:

Ingredient Percent by Weight A commercially available mother liquorbyproduct of dextrose crystallization (Enzose E081) l8.6 Spray driedwhey 2.0 Soy protein (Kaysoy) 2.0 Com steep liquor 2.5 Monopotasiumphosphate 0.4 Calcium carbonate 0.15 Magnesium sulfate 0.15 Lard oil 0.33 Water balance Additional Enzose E0 81 ingredient is added periodicallyto maintain reducing sugar level in the mash between 0.4 and 1 percentby weight during fermentation.

What is claimed is:

1. The process of culturing the micro-organism Bacillus subtilis,variety licheniformis, NRRL B3723 in a nutrient medium, and thereafterseparating alkaline protease thereby produced from said medium.

2. The process of claim 1 in which said micro-organism is culturedaerobically in a nutrient medium at a temperature of 30 C. and pH of5.0to 8.0.

3. The process of claim 2 in which said micro-organism if cultured at atemperature of 34 to 38 C., and a pH of 6.0 to 6.6.

4. The process of claim 2 in which said protease is separated byfiltering the product of said culturing and thereafter precipitating theprotease from the filtrate with an alcohol having no more than threecarbon atoms.

5. The process of claim 4 in which said alcohol is methanol.

6. The process of claim 2 in which said nutrient medium contains from 2to 5 weight percent of cottonseed protein.

7. The process of culturing a micro-organism of the species Bacillussubtilis, variety licheniformis aerobically in a nutrient medium at a pHof 5.0 to 8.0 in which said nutrient medium contains from 2 to 5 weightpercent of cottonseed protein, to obtain an improved yield of alkalineprotease.

8. The process of claim 7 in which said temperature is 30 to 40 C.

9. The process of claim 8 in which said micro-organism is cultured at atemperature of 34 to 38 C. and a pH of 6.0 to 6.6.

10. The process of claim 7 in which said micro-organism is Bacillussubtilis, variety licheniformis, NRRL B3723.

2. The process of claim 1 in which said micro-organism is culturedaerobically in a nutrient medium at a temperature of 30* C. and pH of5.0 to 8.0.
 3. The process of claim 2 in which said micro-organism ifcultured at a temperature of 34* to 38* C., and a pH of 6.0 to 6.6. 4.The process of claim 2 in which said protease is separated by filteringthe product of said culturing and thereafter precipitating the proteasefrom the filtrate with an alcohol having no more than three carbonatoms.
 5. The process of claim 4 in which said alcohol is methanol. 6.The process of claim 2 in which said nutrient medium contains from 2 to5 weight percent of cottonseed protein.
 7. The process of culturing amicro-organism of the species Bacillus subtilis, variety licheniformisaerobically in a nutrient medium at a pH of 5.0 to 8.0 in which saidnutrient medium contains from 2 to 5 weight percent of cottonseedprotein, to obtain an improved yield of alkaline protease.
 8. Theprocess of claim 7 in which said temperature is 30* to 40* C.
 9. Theprocess of claim 8 in which said micro-organism is cultured at atemperature of 34* to 38* C. and a pH of 6.0 to 6.6.
 10. The process ofclaim 7 in which said micro-organism is Bacillus subtilis, varietylicheniformis, NRRL B3723.